"I am adhering to HIV treatment so that I can live to support her": A qualitative study of upward intergenerational support in South Africa.

Background: Intergenerational family care, which was upended by the HIV epidemic in sub-Saharan Africa (SSA), may return to a pre-HIV era arrangement as access to antiretroviral therapy (ART) expands and treated adults can once again provide support for older household members. Empirical research has demonstrated positive 'spillover effects' of ART uptake from treated adults to younger generations, yet much less is known about the nature and breadth of such effects to older generations. This study explores the role and lived experiences among adults who take up ART and those of an older generation with whom they live. Methods: We conducted a qualitative study consisting of semi-structured interviews (n = 46) embedded in the Agincourt Health and Demographic Surveillance System (HDSS) in rural South Africa, between July and September 2022. We purposefully sampled two respondent categories: (i) young or middle-aged adults on ART (aged 18-59 years old); and (ii) older adults (aged ≥60 years old) who were affiliated with a young or middle-aged adult on ART. We used thematic content analysis to extract, code, and categorise relevant text by types of upward spillover effects from ART in younger adults to older adults. Quantitative data was extracted from the existing Agincourt HDSS database and matched to qualitative interview data based on Clinic link unique identifiers of study participants. Results: Mean age was 41 years among young or middle-aged adults (n = 29) and 72 years among older adults (n = 17). Among younger adults, time on ART ranged from five months to more than 21 years. Both young or middle-aged adults on ART and older adults reported positive spillover effects for older adults across five main tiers: caregiving, financial support, physical and mental health, living arrangements and household relationships, and stigma and reputation. Spillover challenges included financial costs and caregiving responsibilities following ART initiation of young or middle-aged adults, although these additional caregiving responsibilities were generally not perceived as particularly burdensome. Conclusions: ART is likely to benefit older adults in South Africa whose families are affected by HIV. This study identified a wide range of perceived spillover effects from ART in younger adults to older adults, including improvements to upward intergenerational support. These qualitative findings offer a guide to researchers, policymakers, and donors to capitalise on the broader societal effects of a large-scale health intervention to further support family structures and meet the needs of a growing older population.

The effect of school-entry age on health is understudied in low- and middle-income countries: A scoping review and future directions for research.

Background: Substantive literature has assessed the impact of starting school at younger ages relative to peers on health in high-income countries (HICs), but there is little evidence from low- and middle-income countries (LMICs). Conclusions drawn from HICs may not apply to different education contexts and health threats. This study maps the empirical evidence on the effect of school-entry age on health in LMICs and identifies directions for future research. Methods: We conducted a scoping review between August and September 2022 by systematically searching the health sciences, education, economics, psychology, and general sciences literature and included quantitative and qualitative studies. The exposure of interest was relative age for grade defined as starting or progressing through school at a younger or older age compared to peers who are in the same grade. We extracted key characteristics of included studies and summarized their findings. We categorized results into broad health domains which emerged a posteriori from our analyses of included studies, including neurodevelopment and mental health, sexual and reproductive health, non-communicable diseases, and nutrition. Findings: We identified 8 studies from middle-income countries published between 2017 and 2022. Among those studies, we identified 3 quasi-experimental studies using data from Brazil, Mexico, and Vietnam, and 5 observational studies primarily from Türkiye. Children starting school earlier had an increased risk of being diagnosed with attention deficit hyperactivity disorder, earlier sexual debut and cohabitation, adolescent pregnancy, adolescent marriage, and engaged more frequently in risky behavior compared to children who started school later. Pregnant women who started school younger also had fewer prenatal care visits and experienced more pregnancy complications. Although most studies identified negative health consequences from starting school earlier, the evidence for nutritional outcomes, such as overweight and stunting, was mixed. No studies were identified from low-income countries. Conclusions: Little is known about the health consequences of school-entry age in low-resource settings. Additional research is needed to investigate the impact of relative age for grade, whether and how these effects persist into adulthood, and to inform strategies that can offset potential disadvantages stemming from school-entry cut-off dates.

Intergenerational spillover effects of antiretroviral therapy in sub-Saharan Africa: a scoping review and future directions for research.

BACKGROUND: Antiretroviral therapy (ART) may influence individuals who do not receive the intervention but who are connected in some way to the person who does. Relatively little is known, however, about the size and scope of, what we term, spillover effects of ART. We explored intergenerational spillover effects of ART in sub-Saharan Africa (SSA) and identified several directions for future research. METHODS: We conducted a scoping review between March and April 2022. We systematically searched PubMed, PsycINFO, EconLit, OTseeker, AIDSInfo, Web of Science, CINHAL, Google Scholar and African Index Medicus. We analysed the distribution of included studies over time and summarised their findings. We examined the intergenerational impact of ART provision to working-age adults living with HIV on children ('downward' spillover effects) and older adults ('upward' spillover effects). We categorised types of intergenerational spillover effects according to broad themes which emerged from our analysis of included studies. FINDINGS: We identified 26 studies published between 2005 and 2022 with 16 studies assessing spillover effects from adults to children (downward), and 1 study explicitly assessing spillover effects from working-age adults to older adults (upward). The remaining studies did not fully specify the direction of spillover effects. Most spillover effects of ART to household and family members were beneficial and included improvements in wealth, labour market outcomes, health outcomes and health services utilisation, schooling, and household composition. Both children and older adults benefited from ART availability among adults. Detrimental spillover effects were only reported in three studies and included financial and opportunity costs associated with health services utilisation and food insecurity in the first year after ART. CONCLUSIONS: ART may lead to substantial spillover effects across generations and sectors in SSA. Further research is needed to capitalise on positive spillover effects while mitigating potential negative spillover effects. The returns to investments in large-scale health interventions such as ART may be underestimated without considering these societal benefits.

Metal Exposure and Sex Shape the Fatty Acid Profile of Midges and Reduce the Aquatic Subsidy to Terrestrial Food Webs.

Aquatic micropollutants can be transported to terrestrial systems and their consumers by emergent aquatic insects. However, micropollutants, such as metals, may also affect the flux of physiologically important polyunsaturated fatty acids (PUFAs). As certain PUFAs have been linked to physiological fitness and breeding success of terrestrial consumers, reduced fluxes from aquatic systems could affect terrestrial populations and food webs. We chronically exposed larvae of the aquatic insect Chironomus riparius to a range of environmentally relevant sediment contents of cadmium (Cd) or copper (Cu) in a 28-day microcosm study. Since elevated water temperatures can enhance metals' toxic effects, we used two temperature regimes, control and periodically elevated temperatures (heat waves) reflecting an aspect of climate change. Cd and Cu significantly reduced adult emergence by up to 95% and 45%, respectively, while elevated temperatures had negligible effects. Both metal contents were strongly reduced (∼90%) during metamorphosis. Furthermore, the chironomid FA profile was significantly altered during metamorphosis with the factors sex and metal exposure being relevant predictors. Consequently, fluxes of physiologically important PUFAs by emergent adults were reduced by up to ∼80%. Our results suggest that considering fluxes of physiologically important compounds, such as PUFAs, by emergent aquatic insects is important to understand the implications of aquatic micropollutants on aquatic-terrestrial meta-ecosystems.

Model-based signal tracking in the quantitative analysis of time series of NMR spectra.

Hard modeling of NMR spectra by Gauss-Lorentz peak models is an effective way for dimensionality reduction. In this manner high-dimensional measured data are reduced to low-dimensional information as peak centers, amplitudes or peak widths. For time series of spectra these parameters can be assumed to be smooth functions in time. We suggest to model these time-dependent parameter functions by cubic spline functions, which makes a stable quantitative analysis of NMR series possible even for crossing, highly overlapping peaks. Applications are presented for the batch distillation of methanol and diethylamine, and the reaction of acetic anhydride with 2-propanol.

Insights into the translational and rotational dynamics of cations and anions in protic ionic liquids by means of NMR fast-field-cycling relaxometry.

Understanding the translational and rotational dynamics of cations and anions in hydrogen bonded protic ionic liquids (PIls) is still a challenge. In this study, we determine self-diffusion coefficients and rotational correlation times of both ions in triethylammonium based PILs by means of NMR Fast-Field-Cycling (FFC) relaxometry. Global fits of 1H and 19F nuclear magnetic relaxation dispersion (NMRD) curves allowed proper separation into intra and inter molecular relaxation rates for both NMR sensitive nuclei and thus a reliable description of translational and rotational motion for both ions individually. The diffusion coefficients of the cations are in the order of 6 × 10-11 m2 s-1 at room temperature and about 50 per cent larger than those of the anions. The diffusion coefficients of cations and anions in both PILs were compared with those we derived from applying an universal dispersion power law and those known from pulsed field gradient (PFG) NMR studies. Considering the Nernst-Einstein equation, molar conductivities were calculated from cationic and anionic diffusion coefficients and related to directly measured molar conductivities, allowing the determination of the degree of dissociation. The rotational correlation times τR ranging from 50 ps up to 2 ns as a function of temperature were compared with those obtained from high-field NMR quadrupolar relaxation time measurements addressing explicitly the rotation of the NH vector and giving insights into the acidic proton mobility. The Stokes-Einstein and Stokes-Einstein-Debye relations were applied to relate the diffusion coefficients and rotational correlation times to the macroscopic bulk viscosity. The results were also discussed with respect to the archetypical PIL ethylammonium nitrate.

Reaction rate ambiguities for perturbed spectroscopic data: Theory and implementation.

The analysis of reaction systems and their kinetic modeling is important for both exploratory research and process design. Multivariate curve resolution (MCR) methods are state-of-the-art tools for the analysis of spectral series, but are also affected by an unavoidable solution ambiguity that impacts the obtained concentration profiles, spectra and model parameters. These uncertainties depend on the underlying model and the magnitude of the measurement perturbations. We present a general theoretical approach together with a computational method for the analysis of the solution ambiguity underlying arbitrary kinetic models. The main idea is to determine all those model parameters for which the corresponding pure component factorizations satisfy all given constraints within small error tolerances. This makes it possible to determine bands of concentration profiles and spectra that reflect the underlying ambiguity and circumscribes the potential reliability of MCR solutions. False conclusions on the uniqueness of a solution can be prevented. The procedure can be applied as a post-processing step to MCR methods as MCR-ALS, ReactLab or others. The Matlab program code is freely accessible and includes not only the proposed ambiguity analysis but also an MCR hard-modeling approach. Application studies are presented for two experimental data sets, namely for UV/Vis spectra on the relaxation of a photoexcited state of benzophenone and for Raman spectra on an aldehyde formation process.

UV Sensor Based on Fiber Bragg Grating Covered with Graphene Oxide Embedded in Composite Materials.

Polymer-matrix composites degrade under the influence of UV radiation in the range of the 290-400 nm band. The degradation of polymer-matrix composites exposed to UV radiation is characterized by extensive aging of the epoxy matrix, resulting in deterioration of their mechanical properties. Glass fibers/epoxy resin composites were made by an out-of-autoclave method whereas a fiber optic sensor was placed between different layers of laminates. In our work, we used a fiber Bragg grating sensor covered with graphene oxide and embedded in a polymer matrix composite to monitor UV radiation intensity. Measurements of UV radiation may allow monitoring the aging process of individual components of the polymer composite. In order to estimate the number of microcracks of epoxy resin, microstructure observations were carried out using a scanning electron microscope.

N-acetylcysteine Provides Cytoprotection in Murine Oligodendrocytes through Heme Oxygenase-1 Activity.

Oligodendrocytic injury by oxidative stress can lead to demyelination, contributing to neurodegeneration. We investigated the mechanisms by which an antioxidant, N-acetylcysteine (NAC), reduces oxidative stress in murine oligodendrocytes. We used normal 158N and mutant 158JP cells with endogenously high reactive oxygen species (ROS) levels. Oxidative stress was induced in 158N cells using hydrogen peroxide (H2O2, 500 µM), and both cells were treated with NAC (50 µM to 500 µM). ROS production, total glutathione (GSH) and cell survival were measured 24 h after treatment. In normal cells, H2O2 treatment resulted in a ~5.5-fold increase in ROS and ~50% cell death. These deleterious effects of oxidative stress were attenuated by NAC, resulting in improved cell survival. Similarly, NAC treatment resulted in decreased ROS levels in 158JP cells. Characterization of mechanisms underlying cytoprotection in both cell lines revealed an increase in GSH levels by NAC, which was partially blocked by an inhibitor of GSH synthesis. Interestingly, we observed heme oxygenase-1 (HO-1), a cytoprotective enzyme, play a critical role in cytoprotection. Inhibition of HO-1 activity abolished the cytoprotective effect of NAC with a corresponding decrease in total antioxidant capacity. Our results indicate that NAC promotes oligodendrocyte survival in oxidative stress-related conditions through multiple pathways.

Facile Synthesis of a Stable Side-on Phosphinyne Complex by Redox Driven Intramolecular Cyclisation.

Alkyne complexes with vicinal substitution by a Lewis acid and a Lewis base at the coordinated alkyne are prospective frustrated Lewis pairs exhibiting a particular mutual distance and, hence, a specific activation potential. In this contribution, investigations on the generation of a WII alkyne complex bearing a phosphine as Lewis base and a carbenium group as Lewis acid are presented. Independently on potential substrates added, an intramolecular cyclisation product was always isolated. A subsequent deprotonation step led to an unprecedented side-on λ5 -phosphinyne complex, which is interpreted as highly zwitterionic according to visible absorption spectroscopy supported by TD-DFT. Low-temperature 31 P NMR and EPR spectroscopic measurements combined with time-dependent IR-spectroscopic monitoring provided insights in the mechanism of the cyclisation reaction. Decomposition of the multicomponent IR spectra by multivariate curve resolution and a kinetic hard-modelling approach allowed the derivation of kinetic parameters. Assignment of the individual IR spectra to potential intermediates was provided by DFT calculations.

Antiretroviral therapy coverage associated with increased co-residence between older and working-age adults in Africa.

OBJECTIVES: To determine whether national antiretroviral therapy (ART) coverage is associated with changes in the living arrangements of older adults. DESIGN: Retrospective analysis using 103 nationally representative surveys from 28 African countries between 1991 and 2015. METHODS: The sample consisted of individuals aged at least 60 years. We investigated how three measures of living arrangements of older adults have changed with ART coverage: the number of older individuals living without working-age adults, the number of older individuals living with only dependent children (i.e. 'missing generation' households), and the number of working-age adults per household where an older individual lives. RESULTS: Our sample consisted of 297 331 older adults. An increase in ART coverage of 1% was associated with a 0.7 percentage point reduction (P < 0.001) in the probability of an older adult living without working-age adult and a 0.2 percentage point reduction (P = 0.005) in the probability of an older adult living in a 'missing generation' household. Increases in ART coverage were also associated with more working-age adults in households with at least one older adult. In our study countries, representing 75% (749 million) of the sub-Saharan population, an additional 103 000-358 000 older adults could be living with working-age adults as a result of increased ART coverage (1%). CONCLUSION: The scale-up of ART has likely led to substantial increases in co-residence between older and working-age adults in Africa. Returns to investments in HIV treatment will be too low, if the social benefits from these changes in living arrangements of older adults are not taken into account.

Multi-objective optimization for an automated and simultaneous phase and baseline correction of NMR spectral data.

Spectral data preprocessing is an integral and sometimes inevitable part of chemometric analyses. For Nuclear Magnetic Resonance (NMR) spectra a possible first preprocessing step is a phase correction which is applied to the Fourier transformed free induction decay (FID) signal. This preprocessing step can be followed by a separate baseline correction step. Especially if series of high-resolution spectra are considered, then automated and computationally fast preprocessing routines are desirable. A new method is suggested that applies the phase and the baseline corrections simultaneously in an automated form without manual input, which distinguishes this work from other approaches. The underlying multi-objective optimization or Pareto optimization provides improved results compared to consecutively applied correction steps. The optimization process uses an objective function which applies strong penalty constraints and weaker regularization conditions. The new method includes an approach for the detection of zero baseline regions. The baseline correction uses a modified Whittaker smoother. The functionality of the new method is demonstrated for experimental NMR spectra. The results are verified against gravimetric data. The method is compared to alternative preprocessing tools. Additionally, the simultaneous correction method is compared to a consecutive application of the two correction steps.

A chemometric study in the area of feasible solution of an acid-base titration of N-methyl-6-oxyquinolone.

Multivariate curve resolution methods aim at recovering the underlying chemical components from spectroscopic data on chemical reaction systems. In most cases the spectra and concentration profiles of the pure components cannot be uniquely determined from the given spectral data. Instead continua of possible factors exist. This fact is known as rotational ambiguity. The sets of all possible pure component factors can be represented in the so-called area of feasible solutions (AFS). This paper presents an AFS study of the pure component reconstruction problem for a series of UV/Vis spectra taken from an acid-base titration of N-methyl-6-oxyquinolone. Additional information on the equilibrium concentration profiles for a varying acid concentration is taken from fluorescence measurements. On this basis chemometric duality arguments lead to the construction of a unique final solution.

Viewing the Valence Electronic Structure of Ferric and Ferrous Hexacyanide in Solution from the Fe and Cyanide Perspectives.

The valence-excited states of ferric and ferrous hexacyanide ions in aqueous solution were mapped by resonant inelastic X-ray scattering (RIXS) at the Fe L2,3 and N K edges. Probing of both the central Fe and the ligand N atoms enabled identification of the metal- and ligand-centered excited states, as well as ligand-to-metal and metal-to-ligand charge-transfer excited states. Ab initio calculations utilizing the RASPT2 method were used to simulate the Fe L2,3-edge RIXS spectra and enabled quantification of the covalencies of both occupied and empty orbitals of π and σ symmetry. We found that π back-donation in the ferric complex is smaller than that in the ferrous complex. This is evidenced by the relative amounts of Fe 3d character in the nominally 2π CN(-) molecular orbital of 7% and 9% in ferric and ferrous hexacyanide, respectively. Utilizing the direct sensitivity of Fe L3-edge RIXS to the Fe 3d character in the occupied molecular orbitals, we also found that the donation interactions are dominated by σ bonding. The latter was found to be stronger in the ferric complex, with an Fe 3d contribution to the nominally 5σ CN(-) molecular orbitals of 29% compared to 20% in the ferrous complex. These results are consistent with the notion that a higher charge at the central metal atom increases donation and decreases back-donation.

On the ambiguity of the reaction rate constants in multivariate curve resolution for reversible first-order reaction systems.

If for a chemical reaction with a known reaction mechanism the concentration profiles are accessible only for certain species, e.g. only for the main product, then often the reaction rate constants cannot uniquely be determined from the concentration data. This is a well-known fact which includes the so-called slow-fast ambiguity. This work combines the question of unique or non-unique reaction rate constants with factor analytic methods of chemometrics. The idea is to reduce the rotational ambiguity of pure component factorizations by considering only those concentration factors which are possible solutions of the kinetic equations for a properly adapted set of reaction rate constants. The resulting set of reaction rate constants corresponds to those solutions of the rate equations which appear as feasible factors in a pure component factorization. The new analysis of the ambiguity of reaction rate constants extends recent research activities on the Area of Feasible Solutions (AFS). The consistency with a given chemical reaction scheme is shown to be a valuable tool in order to reduce the AFS. The new methods are applied to model and experimental data.

Mechanisms of Antioxidant Induction with High-Dose N-Acetylcysteine in Childhood Cerebral Adrenoleukodystrophy.

BACKGROUND: Childhood cerebral adrenoleukodystrophy (CCALD), a progressive demyelinating disease affecting school-aged boys, causes death within a few years. Oxidative stress is an important contributing factor. N-acetylcysteine (NAC; 280 mg/kg/day) added as adjunctive therapy to reduced-intensity hematopoietic cell transplantation (HCT) improves survival in advanced cases. However, the mechanisms underlying the benefits of NAC are unclear. OBJECTIVE: The aim of this study was to understand the mechanism of action of NAC in the setting of HCT in CCALD. METHODS: Immunoassays were carried out to determine changes in heme oxygenase-1 (HO-1) and ferritin expression in plasma samples collected from boys with CCALD at three different timepoints during the course of transplantation. In addition, the induction of HO-1 was also confirmed in normal fibroblasts following incubation with 10-100 µmol/L NAC for 4 h. RESULTS: Following NAC therapy we observed an increase in expression of the antioxidants HO-1 (~4-fold) and its effector ferritin (~160-fold) in patient samples as compared with baseline. We also observed that NAC exposure significantly increased HO-1 expression in fibroblasts. CONCLUSION: Our data suggest that HO-1 is a possible target protein of NAC and a mediator of its cytoprotective effects in these patients.

Display glass for low-loss and high-density optical interconnects in electro-optical circuit boards with eight optical layers.

Parallel optical interconnects on-board level requires low propagation loss in wavelength range between 850 and 1550 nm to be compatible with datacom and telecom optical engines. For highest integration density tight waveguide bends and a scalable number of optical layers should be manufacturable for 2D interfaces to optical fiber array connectors and photonic assembly I/O's. We developed a glass waveguide panel process for double-sided processing of commercial available display glass by applying a two-step thermal ion-exchange process for low-loss multi-mode graded-index waveguides. Multiple glass waveguide panels can be embedded between electrical layers. The generic concept enables fabrication of high-density integration (HDI) electro-optical circuit boards (EOCB) with high number of optical and electrical layers. Waveguides with high NA of 0.3 for low bend losses could be achieved in glass with propagation loss of 0.05 dB/cm for all key wavelengths. Four of those glass waveguide panels were embedded in an EOCB demonstrator with size of 280 x 233 mm² providing eight optical layers with 96 channels in an area of 2.8 x 1.5 mm². To the best of our knowledge it's the highest number of layers that has ever been demonstrated for an EOCB.

Aspirin increases nitric oxide formation in chronic stable coronary disease.

INTRODUCTION: There are no published randomized data on secondary prevention in humans about whether aspirin affects nitric oxide (NO) formation. In patients with chronic stable coronary disease, we tested whether aspirin at clinically relevant doses increases NO formation. MATERIALS AND METHODS: In a randomized, double-blind trial, 37 patients from 2 cardiology office practices were assigned to daily doses of 81, 162.5, 325, 650, or 1300 aspirin for 12 weeks. Primary prespecified outcome measures were changes in heme oxygenase (HO-1), a downstream target of NO formation, and asymmetrical dimethyl arginine (ADMA), a competitive inhibitor of NO synthase. RESULTS: There were no significant differences for HO-1 or ADMA between any of the clinically relevant doses of aspirin tested, so all were combined. For HO-1, there was a significant increase (10.29 ± 2.44, P < .001) from baseline (15.37 ± 1.85) to week 12 (25.66 ± 1.57). The mean ratio (MR) of week 12 to baseline for HO-1 was significantly higher than 1.0 (1.67, confidence interval [CI] from 1.60 to 1.74, P < .001). For ADMA, there was a significant decrease (-0.24 ± 0.11, P < .001) from baseline (0.78 ± 0.08) to week 12 (0.54 ± 0.07). The MR of week 12 to baseline for ADMA was significantly lower than 1.0 (0.69, CI from 0.66 to 0.73, P < .001). CONCLUSIONS: In patients with chronic stable coronary disease, all clinically relevant daily doses of aspirin tested, from 81 to 1300 mg, produce similar and statistically significant increases in HO-1 and decreases in ADMA. These are the first randomized data on secondary prevention patients. These data support the hypothesis that aspirin has additional beneficial effects mediated through NO formation. Further research, including direct randomized comparisons on atherosclerosis using noninvasive techniques as well as on occlusive vascular disease events, is necessary.

L-Edge X-ray Absorption Spectroscopy of Dilute Systems Relevant to Metalloproteins Using an X-ray Free-Electron Laser.

L-edge spectroscopy of 3d transition metals provides important electronic structure information and has been used in many fields. However, the use of this method for studying dilute aqueous systems, such as metalloenzymes, has not been prevalent because of severe radiation damage and the lack of suitable detection systems. Here we present spectra from a dilute Mn aqueous solution using a high-transmission zone-plate spectrometer at the Linac Coherent Light Source (LCLS). The spectrometer has been optimized for discriminating the Mn L-edge signal from the overwhelming O K-edge background that arises from water and protein itself, and the ultrashort LCLS X-ray pulses can outrun X-ray induced damage. We show that the deviations of the partial-fluorescence yield-detected spectra from the true absorption can be well modeled using the state-dependence of the fluorescence yield, and discuss implications for the application of our concept to biological samples.

Enhanced detection of hydrogen sulfide generated in cell culture using an agar trap method.

Lack of reliable methods to accurately measure hydrogen sulfide (H(2)S) produced in vitro has impeded research on the physiology of this gaseous mediator. Current in vitro methods involve measurement of H(2)S in cell culture media following incubation with H(2)S-releasing compounds. However, this method is inaccurate because H(2)S gas has a short life and thus evades detection. To overcome this, we have adapted a method that employs a modified agar layer to instantly trap H(2)S, allowing measurement of H(2)S accumulated with time. The amount of H(2)S trapped in the agar is quantified using an in situ methylene blue assay. We were able to detect H(2)S produced from sodium hydrogen sulfide (NaHS) added at concentrations as low as 10 µM. Following a 24-h incubation of endothelial-like or vascular smooth muscle cells with 50 µM NaHS, we were able to recover twice more H(2)S than conventional methods. When H(2)S-releasing compounds L-cysteine and N-acetylcysteine were added to the cell culture, the amount of H(2)S increased in a concentration-, time-, and cell line-dependent manner. In conclusion, we have developed an improved method to quantify H(2)S generated in vitro. This method could be used to screen compounds to identify potential H(2)S donors and inhibitors for therapeutic use.